Dawn E. W. Livingstone

Intra-adipose sex steroid metabolism and body fat distribution in idiopathic human obesity

Objective  Causes of visceral fat accumulation include glucocorticoid excess or decreased oestrogen/androgen ratio either in plasma or within adipose tissue. In obese subjects, the intra‐adipose cortisol‐generating enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) is increased, but information on sex steroid signalling is sparse. We aimed to test associations between body fat or fat distribution and mRNA transcript levels for androgen and oestrogen receptors and for enzymes metabolizing sex steroids in adipose tissue.

Reconciling the nutritional and glucocorticoid hypotheses of fetal programming

Fetal growth restriction associates with increased risk of adult cardiometabolic and neuropsychiatric disorders. Both maternal malnutrition [notably a low‐protein (LP) diet] and stress/glucocorticoid exposure reduce fetal growth and cause persisting abnormalities (programming) in adult offspring. Deficiency of placental 11 β‐hydroxysteroid dehydrogenase‐2 (11β‐HSD2), which inactivates glucocorticoids, is reduced by an LP diet and has been proposed as a unifying mechanism. Here, we explored the importance of glucocorticoids and placental 11β‐HSD2 in dietary programming. Pregnant mice were fed a control or isocaloric LP diet throughout gestation. The LP diet first elevated fetal glucocorticoid levels, then reduced placental growth, and finally decreased fetal weight near term by 17%. Whereas the LP diet reduced placental 11β‐HSD2 activity near term by ~25%, consistent with previous reports, activity was increased between 20 and 40% at earlier ages, implying that glucocorticoid overexposure in LP fetuses occurs via 11β‐HSD2‐independent mechanisms. Consistent with this, heterozygous 11β‐HSD2+/‐ crosses showed that although both LP and 11β‐HSD2 deficiency reduced fetal growth, LP indeed acted independently of 11β‐HSD2. Instead, the LP diet induced the fetal hypothalamic‐pituitary‐adrenal axis per se. Thus, maternal malnutrition and placental 11β‐HSD2 deficiency act via distinct processes to retard fetal growth, both involving fetoplacental overexposure to glucocorticoids but from distinct sources.—Cottrell, E. C., Holmes, M. C., Livingstone, D. E., Keynon, C. J., Seckl, J. R. Reconciling the nutritional and glucocorticoid hypotheses of fetal programming. FASEB J. 26, 1866‐1874 (2012). www.fasebj.org

Bile acids modulate glucocorticoid metabolism and the hypothalamic–pituitary–adrenal axis in obstructive jaundice ☆

Background & Aims Suppression of the hypothalamic–pituitary–adrenal axis occurs in cirrhosis and cholestasis and is associated with increased concentrations of bile acids. We investigated whether this was mediated through bile acids acting to impair steroid clearance by inhibiting glucocorticoid metabolism by 5β-reductase. Methods The effect of bile acids on glucocorticoid metabolism was studied in vitro in hepatic subcellular fractions and hepatoma cells, allowing quantitation of the kinetics and transcript abundance of 5β-reductase. Metabolism was subsequently examined in vivo in rats following dietary manipulation or bile duct ligation. Finally, glucocorticoid metabolism was assessed in humans with obstructive jaundice. Results In rat hepatic cytosol, chenodeoxycholic acid competitively inhibited 5β-reductase (Ki 9.19 ± 0.40 μM) and reduced its transcript abundance (in H4iiE cells) and promoter activity (reporter system, HepG2 cells). In Wistar rats, dietary chenodeoxycholic acid (1% w/w chow) inhibited hepatic 5β-reductase activity, reduced urinary excretion of 3α,5β-tetrahydrocorticosterone and reduced adrenal weight. Conversely, a fat-free diet suppressed bile acid levels and increased hepatic 5β-reductase activity, supplementation of the fat-free diet with CDCA reduced 5β-reductase activity, and urinary 3α,5β-reduced corticosterone. Cholestasis in rats suppressed hepatic 5β-reductase activity and transcript abundance. In eight women with obstructive jaundice, relative urinary excretion of 3α,5β-tetrahydrocortisol was significantly lower than in healthy controls. Conclusion These data suggest a novel role for bile acids in inhibiting hepatic glucocorticoid clearance, of sufficient magnitude to suppress hypothalamic–pituitary–adrenal axis activity. Elevated hepatic bile acids may account for adrenal insufficiency in liver disease.

Salicylate Downregulates 11β-HSD1 Expression in Adipose Tissue in Obese Mice and in Humans, Mediating Insulin Sensitization

Recent trials show salicylates improve glycemic control in type 2 diabetes, but the mechanism is poorly understood. Expression of the glucocorticoid-generating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in adipose tissue is increased in vitro by proinflammatory cytokines and upregulated in obesity. 11β-HSD1 inhibition enhances insulin sensitivity. We hypothesized that salicylates downregulate 11β-HSD1 expression, contributing to their metabolic efficacy. We treated diet-induced obese (DIO) 11β-HSD1–deficient mice and C57Bl/6 mice with sodium salicylate for 4 weeks. Glucose tolerance was assessed in vivo. Tissue transcript levels were assessed by quantitative PCR and enzyme activity by incubation with 3H-steroid. Two weeks’ administration of salsalate was also investigated in a randomized double-blind placebo-controlled crossover study in 16 men, with measurement of liver 11β-HSD1 activity in vivo and adipose tissue 11β-HSD1 transcript levels ex vivo. In C57Bl/6 DIO mice, salicylate improved glucose tolerance and downregulated 11β-HSD1 mRNA and activity selectively in visceral adipose. DIO 11β-HSD1–deficient mice were resistant to these metabolic effects of salicylate. In men, salsalate reduced 11β-HSD1 expression in subcutaneous adipose, and in vitro salicylate treatment reduced adipocyte 11β-HSD1 expression and induced adiponectin expression only in the presence of 11β-HSD1 substrate. Reduced intra-adipose glucocorticoid regeneration by 11β-HSD1 is a novel mechanism that contributes to the metabolic efficacy of salicylates.

5α-Reductase Type 1 Modulates Insulin Sensitivity in Men

Context: 5α-Reductase (5αR) types 1 and 2 catalyze the A-ring reduction of steroids, including androgens and glucocorticoids. 5α-R inhibitors lower dihydrotestosterone in benign prostatic hyperplasia; finasteride inhibits 5αR2, and dutasteride inhibits both 5αR2 and 5αR1. In rodents, loss of 5αR1 promotes fatty liver. Objective: Our objective was to test the hypothesis that inhibition of 5αR1 causes metabolic dysfunction in humans. Design, Setting, and Participants: This double-blind randomized controlled parallel group study at a clinical research facility included 46 men (20–85 years) studied before and after intervention. Intervention: Oral dutasteride (0.5 mg daily; n = 16), finasteride (5 mg daily; n = 16), or control (tamsulosin; 0.4 mg daily; n = 14) was administered for 3 months. Main Outcome Measure: Glucose disposal was measured during a stepwise hyperinsulinemic-euglycemic clamp. Data are mean (SEM). Results: Dutasteride and finasteride had similar effects on steroid profiles, with reduced urinary androgen and glucocorticoid metabolites and reduced circulating DHT but no change in plasma or salivary cortisol. Dutasteride, but not finasteride, reduced stimulation of glucose disposal by high-dose insulin (dutasteride by −5.7 [3.2] μmol/kg fat-free mass/min, versus finasteride +7.2 [3.0], and tamsulosin +7.0 [2.0]). Dutasteride also reduced suppression of nonesterified fatty acids by insulin and increased body fat (by 1.6% [0.6%]). Glucose production and glycerol turnover were unchanged. Consistent with metabolic effects of dutasteride being mediated in peripheral tissues, mRNA for 5αR1 but not 5αR2 was detected in human adipose tissue. Conclusion: Dual inhibition of 5αRs, but not inhibition of 5αR2 alone, modulates insulin sensitivity in human peripheral tissues rather than liver. This may have important implications for patients prescribed dutasteride for prostatic disease.

Hsd11b2 Haploinsufficiency in Mice Causes Salt Sensitivity of Blood Pressure

Salt sensitivity of blood pressure is an independent risk factor for cardiovascular morbidity. Mechanistically, abnormal mineralocorticoid action and subclinical renal impairment may blunt the natriuretic response to high sodium intake, causing blood pressure to rise. 11&bgr;-Hydroxysteroid dehydrogenase type 2 (11&bgr;HSD2) controls ligand access to the mineralocorticoid receptor, and ablation of the enzyme causes severe hypertension. Polymorphisms in HSD11B2 are associated with salt sensitivity of blood pressure in normotensives. In this study, we used mice heterozygote for a null mutation in Hsd11b2 (Hsd11b2+/−) to define the mechanisms linking reduced enzyme activity to salt sensitivity of blood pressure. A high-sodium diet caused a rapid and sustained increase in blood pressure in Hsd11b2+/− mice but not in wild-type littermates. During the adaptation to high-sodium diet, heterozygotes displayed impaired sodium excretion, a transient positive sodium balance, and hypokalemia. After 21 days of high-sodium feeding, Hsd11b2+/− mice had an increased heart weight. Mineralocorticoid receptor antagonism partially prevented the increase in heart weight but not the increase in blood pressure. Glucocorticoid receptor antagonism prevented the rise in blood pressure. In Hsd11b2+/− mice, high-sodium feeding caused suppression of aldosterone and a moderate but sustained increase in corticosterone. This study demonstrates an inverse relationship among 11&bgr;HSD2 activity, heart weight, and blood pressure in a clinically important context. Reduced activity causes salt sensitivity of blood pressure, but this does not reflect illicit activation of mineralocorticoid receptors by glucocorticoids. Instead, we have identified a novel interaction among 11&bgr;HSD2, dietary salt, and circulating glucocorticoids.

Physiological roles of glucocorticoids during early embryonic development of the zebrafish (Danio rerio)

•  Glucocorticoids are known to be present in the developing zebrafish embryo but little is known about their physiological role at this early stage. •  The zebrafish embryo demonstrates a functional glucocorticoid system from around 48 h post fertilisation. •  This system and the stress response is amenable to pharmacological and genetic manipulation in a manner predicted by mammalian physiology. •  Glucocorticoids play a key developmental role in hatching, swimming and stress response. •  The zebrafish embryo is a relevant model for the study of glucocorticoid physiology.

5α-Reductase type 1 deficiency or inhibition predisposes to insulin resistance, hepatic steatosis and liver fibrosis in rodents

5α-Reductase type 1 (5αR1) catalyses A-ring reduction of androgens and glucocorticoids in liver, potentially influencing hepatic manifestations of the metabolic syndrome. Male mice, homozygous for a disrupted 5αR1 allele (5αR1 knockout [KO] mice), were studied after metabolic (high-fat diet) and fibrotic (carbon tetrachloride [CCl4]) challenge. The effect of the 5α-reductase inhibitor finasteride on metabolism was investigated in male obese Zucker rats. While eating a high-fat diet, male 5αR1-KO mice demonstrated greater mean weight gain (21.6 ± 1.4 vs 16.2 ± 2.4 g), hyperinsulinemia (insulin area under the curve during glucose tolerance test 609 ± 103 vs. 313 ± 66 ng ⋅ mL−1 ⋅ min), and hepatic steatosis (liver triglycerides 136.1 ± 17.0 vs. 89.3 ± 12.1 μmol ⋅ g−1). mRNA transcript profiles in liver were consistent with decreased fatty acid β-oxidation and increased triglyceride storage. 5αR1-KO male mice were more susceptible to fibrosis after CCl4 administration (37% increase in collagen staining). The nonselective 5α-reductase inhibitor finasteride induced hyperinsulinemia and hepatic steatosis (10.6 ± 1.2 vs. 7.0 ± 1.0 μmol ⋅ g−1) in obese male Zucker rats, both intact and castrated. 5αR1 deficiency induces insulin resistance and hepatic steatosis, consistent with the intrahepatic accumulation of glucocorticoids, and predisposes to hepatic fibrosis. Hepatic steatosis is independent of androgens in rats. Variations in 5αR1 activity in obesity and with nonselective 5α-reductase inhibition in men with prostate disease may have important consequences for the onset and progression of metabolic liver disease.

11β-hydroxysteroid dehydrogenase type 1 deficiency in bone marrow-derived cells reduces atherosclerosis

11β‐Hydroxysteroid dehydrogenase type‐1 (11β‐HSD1) converts inert cortisone into active cortisol, amplifying intracellular glucocorticoid action. 11β‐HSD1 deficiency improves cardiovascular risk factors in obesity but exacerbates acute inflammation. To determine the effects of 11β‐HSD1 deficiency on atherosclerosis and its inflammation, atherosclerosis‐prone apolipoprotein E‐knockout (ApoE‐KO) mice were treated with a selective 11β‐HSD1 inhibitor or crossed with 11β‐HSD1‐KO mice to generate double knockouts (DKOs) and challenged with an atherogenic Western diet. 11β‐HSD1 inhibition or deficiency attenuated atherosclerosis (74–76%) without deleterious effects on plaque structure. This occurred without affecting plasma lipids or glucose, suggesting independence from classical metabolic risk factors. KO plaques were not more inflamed and indeed had 36% less T‐cell infiltration, associated with 38% reduced circulating monocyte chemoattractant protein‐1 (MCP‐1) and 36% lower lesional vascular cell adhesion molecule‐1 (VCAM‐1). Bone marrow (BM) cells are key to the atheroprotection, since transplantation of DKO BM to irradiated ApoE‐KO mice reduced atherosclerosis by 51%. 11β‐HSD1‐null macrophages show 76% enhanced cholesterol ester export. Thus, 11β‐HSD1 deficiency reduces atherosclerosis without exaggerated lesional inflammation independent of metabolic risk factors. Selective 11β‐HSD1 inhibitors promise novel antiatherosclerosis effects over and above their benefits for metabolic risk factors via effects on BM cells, plausibly macrophages.—Kipari, T., Hadoke, P. W. F., Iqbal, J., Man, T. Y., Miller, E., Coutinho, A. E., Zhang, Z., Sullivan, K. M., Mitic, T., Livingstone, D. E. W., Schrecker, C., Samuel, K., White, C. I., Bouhlel, M. A., Chinetti‐Gbaguidi, G., Staels, B., Andrew, R., Walker, B. R., Savill, J. S., Chapman, K. E., Seckl, J. R. 11β‐hydroxysteroid dehydrogenase type 1 deficiency in bone marrow‐derived cells reduces atherosclerosis. FASEB J. 27, 1519–1531 (2013). www.fasebj.org

Dysregulation of glucocorticoid metabolism in murine obesity: comparable effects of leptin resistance and deficiency

In obese humans, metabolism of glucocorticoids by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and A-ring reduction (by 5α- and 5β-reductases) is dysregulated in a tissue specific manner. These changes have been recapitulated in leptin resistant obese Zucker rats but were not observed in high-fat fed Wistar rats. Recent data from mouse models suggest that such discrepancies may reflect differences in leptin signalling. We therefore compared glucocorticoid metabolism in murine models of leptin deficiency and resistance. Male ob/ob and db/db mice and their respective littermate controls (n=10–12/group) were studied at the age of 12 weeks. Enzyme activities and mRNA expression were quantified in snap-frozen tissues. The patterns of altered pathways of steroid metabolism in obesity were similar in ob/ob and db/db mice. In liver, 5β-reductase activity and mRNA were increased and 11β-HSD1 decreased in obese mice, whereas 5α-reductase 1 (5αR1) mRNA was not altered. In visceral adipose depots, 5β-reductase was not expressed, 11β-HSD1 activity was increased and 5αR1 mRNA was not altered in obesity. By contrast, in subcutaneous adipose tissue 11β-HSD1 and 5αR1 mRNA were decreased. Systematic differences were not found between ob/ob and db/db murine models of obesity, suggesting that variations in leptin signalling through the short splice variant of the Ob receptor do not contribute to dysregulation of glucocorticoid metabolism.